Human herpesvirus 8 (HHV-8) encodes non-structural proteins that regulate host cell antiviral mechanisms via multiple inhibitory interactions. We have found that viral interferon regulatory factor-1 (vIRF-1) inhibits the pro-apoptotic activity of BH3-only proteins (BOPs) including Bim and Bid by nuclear sequestration (Bim) or directly suppressing their induction of cytochrome c release from mitochondria. In addition, we have found that vIRF-1 can localize to mitochondria independently of its interaction with BOPs and specifically targets mitochondrial detergent-resistant membrane fractions (mitoDRMs), a type of lipid raft. MitoDRMs have been recently identified as a functionally important microdomain of mitochondria and emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. Despite the increasing significance of mitoDRMs, little is known about the role of mitoDRMs in virus infection. To our knowledge, vIRF-1 is the first viral protein known to target to mitoDRMs. Furthermore, our preliminary data have shown that the N-terminal proline-rich domain (PD, 1-75 residues) of vIRF-1 is essential for mitoDRM targeting and protects cells from mitochondrial damage. Also, vIRF-1 co-localizes with mitochondria in an aggregate resembling those occurring during mitophagy, indicating a possible role of vIRF-1 in mitochondria quality control and associated cell survival. To determine the contribution of vIRF-1 through mitoDRM targeting and cell protection to HHV-8 replication, this proposal focuses on examining: 1) the novel structural determinants of mitoDRM targeting by vIRF-1 PD; 2) the molecular mechanisms of vIRF-1 mitoDRM anchoring and cell protection; 3) the functional significance of mitoDRM-localized vIRF-1 in mitophagy and HHV-8 replication. This proposal will thus examine a novel paradigm of virus-host interaction, and potentially provide a basis for future development of novel antiviral agents based on the identified interactions and mechanisms.